The adhesion and activation of resting, circulating platelets at a site of vascular injury is the first step in a process leading to the formation of a thrombus, which is converted into a hemostatic plug. Collagen is one of the major components of the vessel wall responsible for platelet activation. Many types of collagen exist, and seven of these are found in the subendothelial layers. Several different receptors for collagen have been identified on platelets including CD36 (Matsuno, et al., Br. J. Haematol. 92, 960-967, 1996) and a p65 collagen type I specific receptor (Chiang et al., J. Clin. Invest. 100, 514-521, 1997), but the major ones are now considered to be integrin α2β1 and the nonintegrin GPVI. It was determined about 20 years ago that GPVI is a major platelet glycoprotein with a molecular mass in the 60-65-kDa range and an acid pl (Clemetson et al., J. Clin. Invest. 70, 304-311, 1982) which forms a complex together with the Fcγ common subunit. The GPVI subunit contains the collagen binding site and the Fcγ subunit is responsible for signalling. The complex forms one of the major collagen receptors on the platelet surface, critical for platelet activation in response to collagen. Its role as a putative collagen receptor was established following the identification of a patient in Japan with a mild bleeding disorder whose platelets had a specific defect in response to collagen and lacked this receptor (Moroi et al., J. Clin. Invest. 84, 1440-1445, 1989). This patient had also developed autoantibodies to the deficient receptor, and these were used to characterize the molecule further (Sugiyama et al., Blood 69, 1712-1720, 1987). It was also demonstrated that the recognition sequence on collagen for GPVI is a repeated Gly-Pro-Hyp (Hyp=hydroxyproline) triplet within the collagen triple helical structure and that synthetic peptides based on this structure could be used as specific GPVI-directed agonists (Mortonet al., Thromb. Res. 72, 367-372, 1993). The GPVI/FCγ complex was shown to signal to the platelet interior by an immune receptor-like mechanism, involving activation of p72syk and leading by a cascade of kinase/phosphatase/adaptor protein interactions to activation of PLCγ2 and hence to release of granules and platelet aggregation.
Thus, it is clear from the prior art that GPVI-like proteins are very interesting compounds either as tool for the screening of potential agonists or antagonists of the GPVI-collagen interaction or as active principle.
In WO 00/68377 DNA coding for GPVI or biologically fragments thereof, recombinant human GPVI and pharmaceutical compositions thereof are disclosed. Further, the application describes the use of recombinant GPVI as a screening tool for detecting specific inhibitors or activators of platelet-collagen interactions. However, because of its transmembrane domain the recombinant protein is not secreted in the extracellular medium and so it is difficult to purify. Therefore the whole cells expressing this protein have to be used in a screening assay. Further, in view of GPVI as active agent, it is known that recombinant proteins often have a reduced circulating half-life, which leads to a need of frequent application of the drug and therefore results in high costs for the therapy.
Therefore, it was the goal of the present invention to provide molecules with the biological activity of GPVI having the following improved properties: high expression level in the host cell, secretion into the extracellular medium and easy purification, enhanced circulating half-life and, with respect to the use as screening tool, easy detectability by commercially available antibodies.